Project Summary The long-term goal for this project is to develop a highly specific in vivo biosensor to detect and target reversal of apoptosis. Studies of apoptosis have long been based on a general assumption that this cell suicide process is intrinsically irreversible. However, we recently discovered an unexpected cell recovery phenomenon in which dying primary and cancer cells can reverse the apoptotic process even at the execution stage such as caspase-3 activation. We named the reversal of apoptosis ?anastasis?, which means ?rising to life? in Greek. The discovery of anastasis provides new insights into potential therapeutic approaches to intractable diseases. For example, enhancing anastasis to avert apoptosis and help spare injured neurons and heart cells may be beneficial for treating brain injury and heart failure, respectively. Conversely, suppression of anastasis in dying cancer cells may promote cancer cell death and reduce cancer recurrence. However, it is technologically challenging to study anastasis especially in vivo, because the recovered cells are morphologically indistinguishable from the cells that did not attempt apoptosis, and there is no hallmark of anastasis identified yet. Here, we propose to develop in vivo biosensors to track anastasis and to screen for anastasis regulators, using Drosophila melanogaster as a model. In Aim 1, we propose to develop a new generation of anastasis biosensor with high specificity to detect and track anastasis in vivo. We found that apoptotic dying cells can undergo anastasis despite experiencing important checkpoints commonly believed to be the ?point of no return?, such as mitochondrial outer membrane permeabilization (MOMP) and caspase-3 activation. Therefore, we will create an in vivo anastasis biosensor that will permanently label anastatic cells only after they have experienced both MOMP and caspase-3 activation, the two most recognized hallmarks of apoptosis, making this biosensor highly specific to anastasis. To study the molecular mechanism of anastasis, we conducted a time-course gene expression study, and identified multiple genes to display specific up-regulation at the early stage of anastasis. This suggests potential candidates of anastasis regulators. However, the regulatory mechanism of anastasis remains largely unknown, and there is no tool specifically targeting anastatic cells for siRNA screening to the candidate genes. In Aim 2, we will develop an in vivo anastasis biosensor-driven siRNA screening strategy that can knock down the candidate genes specifically in anastatic cells, thereby creating an important tool for identifying the regulators of anastasis. Taken together, these proposed works will create essential tools for future use to study the consequences and mechanisms of anastasis in vivo, laying the foundation for exploring the physiological, pathological, and therapeutic potentials of anastasis.